The overall goal of this project is the identification and molecular characterization of cell surface constituents important in the regulation of mammalian spermatogenesis. Previous studies funded by this grant have developed procedures for the isolation of purified plasma membranes from mouse spermatogenic cells and have provided the first biochemical identifications of a) spermatogenic cell surface marker proteins, b) surface proteins oriented extracellularly and c) con A-binding cell membrane glycoproteins during mouse spermatogenesis. In addition, purified populations of human spermatocytes and spermatids have been prepared. The studies proposed currently represent a continuation of these investigations, concentrating on the analysis of particular polypeptide components detected in spermatocyte and spermatid plasma membranes. Proteins to be studied include, but are not limited to the following: P(a,b), RS(a-d), P greater than 100/6.0, e1 and e2, p 94/5.8, p 53/7.1 and p 55/7/1. Three specific aims are proposed. 1. Monoclonal antibodies will be raised against purified plasma membranes from late mouse spermatogenic cell populations. These antibodies will be characterized by ELISA, immunofluorescent and immunoblot assays to identify the molecular specificity of positive supernatants. Selected reagents will then be used ultrastructurally for the topographical mapping of membrane antigens during spermiogenesis in the mouse. 2. Biochemical experiments include a) the use of insolubilized monoclonal reagents for the preparative isolation of selected germ cell membrane proteins, b) peptide mapping of particular cell surface constituents, c) the analysis of local reorientations within mouse germ cell membranes using hydrophobic molecular probes and d) identification of new marker proteins using hydrophilic molecular probes. 3. Plasma membranes for isolated populations of human spermatocytes and spermatids will be prepared and characterized for direct electrophoretic comparisons with murine material. These investigations represent a unified attempt at the biochemical analysis of developing male germ cell membranes. Antibody preparations and molecular probe analyses will facilitate future physiological assays of membrane function. These studies should also provide the first detailed biochemical data on the membranes of developing human spermatogenic cells other than mature spermatozoa.